We are expanding the range of assay techniques that will allow us to understand the sulfation code in chondroitin sulfate glycosaminoglycan (CS-GAG) chains. These assay techniques take advantage of specific chromatography techniques (ion exchange, hydrophilicity) to separate the different disaccharides and monosaccharides that comprise the GAG chains. This is the only technique capable of doing this. We have also initiated mass spectrometric analysis of these separated GAG chains to begin to determine the sequence of sulfations on the different parts of the GAG chain. Our results indicate that the sulfation pattern of the non-reducing end of the GAG chain is a major determinant of CS signaling. We are conducting studies using a systems biology approach to CSPG actions. We have identified a number of proteins whose phosphorylation is changed rapidly as neurons are exposed to CSPGs. We have confirmed the changes in several of these with Western blotting techniques. We are now validating changes in the several different signal transduction pathways and demonstrating their function in the brain. We have published studies showing that the Nogo receptor and receptor protein tyrosine phosphatase sigma are binding partners for CS GAG chains. In addition our data point to an additional receptor that binds bioactive CSPGs.